Cervical spinal cord hemisection impacts sigh and the respiratory reset in male rats

Abstract Cervical spinal cord injury impacts ventilatory and non‐ventilatory functions of the diaphragm muscle (DIAm) and contributes to clinical morbidity and mortality in the afflicted population. Periodically, integrated brainstem neural circuit activity drives the DIAm to generate a markedly augmented effort or sigh—which plays an important role in preventing atelectasis and thus maintaining lung function. Across species, the general pattern of DIAm efforts during a normal sigh is variable in amplitude and the extent of post‐sigh “apnea” (i.e., the post‐sigh inter‐breath interval). This post‐sigh inter‐breath interval acts as a respiratory reset, following the interruption of regular respiratory rhythm by sigh. We examined the impact of upper cervical (C2) spinal cord hemisection (C2SH) on the transdiaphragmatic pressure (P di) generated during sighs and the post‐sigh respiratory reset in rats. Sighs were identified in P di traces by their characteristic biphasic pattern. We found that C2SH results in a reduction of P di during both eupnea and sighs, and a decrease in the immediate post‐sigh breath interval. These results are consistent with partial removal of descending excitatory synaptic inputs to phrenic motor neurons that results from C2SH. Following cervical spinal cord injury, a reduction in the amplitude of P di during sighs may compromise the maintenance of normal lung function.

There is some contention as to whether the P di generated during sighs in rats has a "stereotypical" amplitude (e.g., >twice eupnea) or biphasic pattern (Khurram et al., 2017;Seven et al., 2018) similar to mice (Pareja-Cajiao et al., 2021), or is variable (e.g., amplitudes <twice eupnea [Khurram et al., 2018]) such as in cats (Reynolds Jr. & Hilgeson, 1965).Additionally, little to no attention has been paid to the postsigh inter-breath interval or respiratory reset in rats.The empirical characterization of sighs in rats would provide a basis for interpreting the neurophysiological underpinnings of DIAm motor unit recruitment, which currently stands as either lower than (Khurram et al., 2017(Khurram et al., , 2018) ) or greater than occlusion P di (Mantilla et al., 2010).The gamut of respiratory neuromotor P di generation in the rat ranges ~10fold from between eupnea (~10-15 cm H 2 O) to P dimax (~100 cm H 2 O).Where sighs fall within this range is currently unknown, as a winnowing of data by inclusion/exclusion criteria obscures interpretation.
The goal of the present study was to examine the impact of C 2 SH on the P di generated during sighs as well as interbreath intervals before and immediately after sighs.We also report the proportion of "stereotypical" sighs meeting common inclusion criteria (i.e., P di amplitudes and a postsigh inter-breath interval of ≥twice eupnea) under normal pre-injury, C 2 SH, and sham (laminectomy) conditions.We hypothesize that C 2 SH will reduce P di amplitude and the duration of the post-sigh inter-breath interval.

| Experimental animals
A total of 16 adult male Sprague-Dawley (SD) rats (~325 g) obtained from Envigo (Indianapolis, IN) were used in the study.We have previously used male rats to examine the impact C 2 SH, with a highly repeatable pathological and physiological outcome (Fogarty et al., 2023;Mantilla, Gransee, et al., 2013;Sieck et al., 2021).Animals were acclimated for at least 7 days prior to any procedure and maintained on an alternating 12:12 h light-dark cycle with ad libitum access to fresh water and food.For all procedures, anesthesia was administered via intraperitoneal injections of xylazine (10 mg/ kg) and ketamine (80 mg/kg).

| Experimental design and timeline
Rats were placed into two experimental groups at random, sham laminectomy, or C 2 SH.These groups were further separated by timepoints into pre-surgery (PRE-SHAM and PRE-C 2 SH) and post-surgery (POST-SHAM and POST-C 2 SH).Anesthetized rats were placed in the prone position, where baseline P di was measures once depth of anesthesia was stable (absence of palpebral and deep pain reflexes).P di measures for both the sham laminectomy and C 2 SH took ~20 min, with the SHAM or C 2 SH surgeries taking ~20-30 min and the final P di recording sessions taking ~20 min.The sham group serves as a time control, as occasional redosing of xylazine (3 mg/kg) and ketamine (25 mg/kg) at ~one third of the initial dose was required (3 in 8 of the C 2 SH and 2 in 8 of the SHAM rats).Thus, all pre-and post-surgery measurements were performed in one experimental session.

| P di measurements
P di was measured as the difference between esophageal and gastric pressures, with two 3.5 French solid-state pressure catheters (SPR-524; Millar Instruments, Houston, TX) inserted through the mouth and into the esophagus or stomach (Fogarty et al., 2023;Khurram et al., 2018;Sieck & Fournier, 1989), both before (uninjured) and after C 2 SH or sham laminectomy.The abdomen was bound to approximate isometric conditions.Esophageal and abdominal pressure signals were recorded and digitized (400 Hz) with PowerLab 4/35, with P di calculated and visualized in real-time with LabChart 9 (ADInstruments, Colorado Springs, CO).From the P di tracings, P di amplitude, respiratory rate (and inter-breath intervals), and inspiratory duration were determined in a manner identical to previous reports (Fogarty et al., 2022(Fogarty et al., , 2023)).All behaviors were recorded with initial O 2 saturation of >90%, in accordance with past studies (Khurram et al., 2019).

| C 2 SH surgery
The surgical methods for C 2 SH have been previously described in detail (Beth Zimmer et al., 2015;Fuller et al., 2006Fuller et al., , 2008;;Miyata et al., 1995;Streeter et al., 2020), with the lesion functionally validated using DIAm EMG (with electrodes implanted 3 days prior to C 2 SH) during the time of surgery and 3 days post-surgery, in accordance with previous studies (Fogarty et al., 2023;Mantilla, Gransee, et al., 2013;Sieck et al., 2021).Briefly, following a dorsal laminectomy, the C 2 spinal cord was cut using a surgical microknife, beginning anterior to the dorsal root entry zone fissure and proceeding ventrally taking care to preserve the dorsal funiculus on the right side, ~7 mm from the midline at the level of the dorsal subdural.The sham group experienced a dorsal laminectomy alone, serving as a time control.

| Statistical analyses
The data sets comprise eupnea (with ~130 individual breaths assessed per rat per surgery status [range: 102-154]), sigh (identified manually as a biphasic breath, with a superimposed additional effort causing a point of inflection at the plateauing inspiratory phase [Cherniack et al., 1981;Reynolds Jr. & Hilgeson, 1965]), and post-sigh eupnea (the breath immediately following a sigh).For sighs and post-sigh breaths, ~8 breaths were analyzed per rat and only during eupneic breathing (range: 5-12), as sigh incidence and amplitudes may be affected by baseline respiratory drive (Jimenez-Ruiz et al., 1985;Wen & Lee, 2018).With eight rats, we were powered (α = 0.05, β = 0.80) to detect a significant difference of >20% in sigh P di amplitude and the eupneic inter-breath intervals following C 2 SH, with mean and variability based on our past study of P di in SD rats (Khurram et al., 2017).Prism 9 was used for all statistical analyses (Graphpad, Carlsbad, CA).We performed paired t-tests, two-or three-way ANOVAs with a Geiser-Greenhouse correction (n.b., we did not assume equal variability between data sets) on all outcome measures.All paired data were done between individual rats pre-surgery (PRE-C 2 SH) and post-C 2 SH (POST-C 2 SH).Similarly, data were paired within behaviors across sham rats pre-surgery (PRE-SHAM) and post laminectomy (POST-SHAM).When appropriate within group differences were evaluated using Bonferroni post hoc tests.When comparing our empirical data generated within this study to the "stereotypical sigh," we are referring to twice eupneic amplitudes and twice eupneic post-sigh inter-breath intervals (Bell et al., 2009;Bell & Haouzi, 2010;Mantilla et al., 2011;Seven et al., 2018).Statistical significance was set at p < 0.05, with p values reported to three significant figures in the results.In the interest or reliability, rigor and robustness of our work, the coefficients of variation of each data set, the relative changes reported (when statistically warranted) and the effect size of each PRE-C 2 SH and POST-C 2 SH comparison are reported in tabular form (Table 1).All data are reported as the mean ± 95% confidence intervals (CI), unless indicated.
Within each rat, when P di amplitude during each motor behavior was expressed as a % of PRE eupnea (P di%PRE ), P di%PRE was dependent on behavior (F = 230.5;p < 0.0001), type of surgery (SHAM vs. C 2 SH; F = 34.7;p = 0.0002), and time (PRE-vs.POST; F = 55.9;p < 0.0001; n = 8 per group, three-way ANOVA; Figure 1c), with P di%PRE reduced POST-C 2 SH compare to PRE-C 2 SH during eupnea (PRE-C 2 SH: 100 ± 0%;  1c).There were no differences between PRE-SHAM and POST-SHAM in P di%PRE during eupnea (p > 0.99), sigh (p > 0.99), and post-sigh (p > 0.99; Bonferroni posttests; Figure 1c).Note that the amplitude of P di for PRE-C 2 SH sighs was ~double the amplitude of P di for PRE-C 2 SH eupnea, whereas POST-C 2 SH sigh P di amplitude was no different compared to PRE-C 2 SH eupnea P di amplitude (p > 0.99; Figure 1c).Overall, these results show the P di amplitudes of eupnea, sigh, and the immediate post-sigh breath were reduced in POST-C 2 SH rats compared to the PRE-C 2 SH condition, with no differences in SHAM time controls.

| Breath durations are unaltered following C 2 SH during eupnea, sigh, and the immediate post-sigh breath
When the mean of each behavior was calculated for each rat, breath duration was dependent on behavior (F = 145.1;p < 0.0001), but not type of surgery (SHAM vs. C 2 SH; F = 0.02; p = 0.787) or time (PRE-vs.POST; F = 1.5; p = 0.227; n = 8 per group, three-way ANOVA; Figure 2a).In all rats, sigh duration was longer compared to eupnea and the post-sigh breath, regardless of pre-or post-injury timepoint (p < 0.05; Bonferroni post-tests).However, there was no difference between C 2 SH and SHAM rats (Figure 2a).
When breath durations were expressed as a % of PRE eupnea, breath duration was dependent on behavior (F = 104.4;p < 0.0001) but not type of surgery (SHAM vs. C 2 SH; F = 0.3; p = 0.469) or time (PRE-vs.POST; F = 1.3; p = 0.270; n = 8 per group, three-way ANOVA; Figure 2b), with sigh having increased durations compared to eupnea and the post-sigh breath in all rats (p < 0.05, Bonferroni post-tests; Figure 2b).Overall, these results show the breath duration of eupnea, sigh, and the immediate postsigh breath was unchanged with C 2 SH or sham.
3.4 | Inter-breath intervals are unaltered following C 2 SH during eupnea, sigh, and the immediate post-sigh breath When the mean of each behavior was calculated for each rat, the inter-breath interval was dependent on behavior (F = 88.9;p < 0.0001), but not type of surgery (SHAM vs. C 2 SH; F = 0.2; p = 0.659) or time (PRE-vs.POST; F = 0.3; p = 0.610; n = 8 per group, three-way ANOVA; Figure 3a).Inter-breath intervals of the post-sigh breath were greater than compared to sigh across all groups (Figure 3a).
When the inter-breath interval was expressed as a % of PRE eupnea, inter-breath interval was dependent on behavior (F = 89.2;p < 0.0001) but not type of surgery (SHAM vs. C 2 SH; F = 0.002; p = 0.924) or time (PRE-vs.POST; F = 0.2; p = 0.661; n = 8 per group, threeway ANOVA; Figure 3b), with greater post-sigh interbreath interval compared to sigh in all groups (p < 0.05, Bonferroni post-tests; Figure 3b).Overall, these results show the inter-breath intervals of eupnea, sigh, and the immediate post-sigh breath were unchanged with C 2 SH or sham.

| Sigh variability and comparisons to stereotypical sigh in C 2 SH rats
As we did not observe a significant effect of time (PRE vs. POST) alone on P di from SHAM rats, we ignored these for the paired analyses of PRE-and POST-C 2 SH rats.Variability across rats was readily apparent in both the PRE-C 2 SH and POST-C 2 SH injury timepoints (Table 1).Within rats, we characterized the % of all sighs within a rat that had P di magnitudes of ≥twice eupnea (i.e., the "stereotypical" sigh amplitude) at PRE-and POST-C 2 SH timepoints (Figure 4a).At the PRE-C 2 SH timepoint, the mean % of sighs within a rat that exhibited stereotypical P di amplitudes was ~48%, ~10 times the % of stereotypical amplitudes observed in the POST-C 2 SH timepoint (~4%; p = 0.0078, Wilcoxon matchedpairs signed-rank test; Figure 4a).
Within rats, we characterized the % of all sighs within a rat that had post-sigh inter-breath intervals of ≥twice eupnea (i.e., the "stereotypical" apneic period) at PRE-and POST-C 2 SH timepoints (Figure 4b).At the PRE-C 2 SH timepoint, the mean % of sighs within a rat that exhibited stereotypical post-sigh inter-breath intervals was ~22%, with no significant difference observed in the POST-C 2 SH timepoint (~35%; p = 0.373, paired t-test; Figure 4b).
The mean duration, inter-breath interval, and amplitudes of the sigh and post-sigh breath were compared to the "stereotypical" sigh at PRE-and POST-C 2 SH timepoints (Figure 5; Table 2), with the reduction in POST-C 2 SH sigh P di amplitude readily observed.Even in the PRE-C 2 SH condition, there was a marked departure of the sigh and post-sigh breath from the "stereotypical" sigh.For sigh amplitudes, only 53% (19/36) from the PRE-C 2 SH and 4% (1/28) from the POST-C 2 SH timepoints satisfied "stereotypical" criteria, with significant differences between PRE-and POST-C 2 SH timepoints (p < 0.0001, Fischer's exact test; Figure 5; Table 2).Likewise, for post-sigh inter-breath intervals, only 22% (8/36) from the PRE-C 2 SH and 32% (9/28) from the POST-C 2 SH timepoints satisfied "stereotypical" criteria, with no differences between timepoints (p = 0.406, Fischer's exact test; Figure 5; Table 2).Incredibly, only 17% of sighs from the PRE-C 2 SH timepoint and not a single sigh from the POST-C 2 SH timepoint satisfied both criteria, with significant differences between PRE-and POST-C 2 SH timepoints (p = 0.031, Fischer's exact test; Figure 5; Table 2).A rather large portion of sighs satisfied neither criterion 25% at PRE-C 2 SH (9/36) and 36% at POST-C 2 SH (10/28) timepoints, with a significant increase between PRE-and POST-C 2 SH timepoints (p = 0.001, Fischer's exact test; Figure 5; Table 2).

| DISCUSSION
This study was the first to characterize the P di generated during sighs and the respiratory reset PRE-and POST-C 2 SH.We present four major findings: (i) P di amplitudes, but not breath durations or inter-breath intervals were altered POST-C 2 SH during eupnea, sigh, and the immediate post-sigh breath; (ii) the amplitudes of P di during sighs were highly variable, with only ~53% of sigh P di amplitudes two times greater than P di during eupnea PRE-C 2 SH, while only ~4% of POST-C 2 SH sighs exhibited P di amplitudes that were > twice eupnea; (iii) while post-sigh inter-breath intervals were longer compared to eupnea, only ~22% of sighs PRE-C 2 SH and ~32% of sighs POST-C 2 SH displayed inter-breath intervals that were twice eupnea; and (iv) overall, PRE-C 2 SH only 17% of all sighs satisfied both "stereotypical" criteria (i.e., amplitudes and inter-breath intervals greater than twice eupnea), while POST-C 2 SH "stereotypical" sighs were absent.These findings have important consequences for the study of respiratory function more generally, and for the effects of C 2 SH and DIAm impairments more specifically.
Sighs have been reported in a myriad of reduced and intact experimental preparations and in a variety of species such as humans (Vlemincx et al., 2010;Wuyts et al., 2011), cats (Cherniack et al., 1981;Orem & Trotter, 1993;van Lunteren et al., 1988), dogs (Katagiri et al., 1998;van Lunteren et al., 1983), rats (Ajayi & Mills, 2017;Bell et al., 2009Bell et al., , 2011;;Bell & Haouzi, 2010;Fogarty et al., 2023;Fuller et al., 2006Fuller et al., , 2008;;Golder et al., 2005;Janssen et al., 2000;Jimenez-Ruiz et al., 1985;Khurram et al., 2017Khurram et al., , 2018;;Seven et al., 2018), and mice (Li et al., 2016;Pareja-Cajiao et al., 2021;Voituron et al., 2010).To date, the focus has been primarily on the pulmonary and EMG characteristics of sigh and the post-sigh breath, with information on rat P di during sigh influenced by the "filtering" effect of inclusion criteria.There is also a total absence of quantitative P di information on the post-sigh breath and whether P di characteristics of sighs are in accordance with plethysmographic results, where duration and tidal volumes are reduced compared to eupnea in cats (Cherniack et al., 1981;Reynolds Jr. & Hilgeson, 1965), but not dogs (van Lunteren et al., 1983).Previously, sigh P di in anesthetized rats was reported to be either less than that generated during airway occlusion in rats (Gill et al., 2015;Khurram et al., 2017Khurram et al., , 2018;;Seven et al., 2018) or greater than occlusion P di (Mantilla et al., 2010).Sigh P di in anesthetized cats was also reported to be less than occlusion P di (Reynolds Jr. & Hilgeson, 1965).In the present study we evaluated augmented breaths with characteristic waveforms without iexclusion criteria of amplitudes or the duration of the post-sigh respiratory reset.We found the mean sigh P di to be ~27 cm H 2 O compared to 14.4 cm H 2 O during eupnea.The mean sigh P di amplitude observed in the present study was moderately higher than that previously reported in Sprague-Dawley rats (Gill et al., 2015;Khurram et al., 2017).The P di generated during airway occlusion in anesthetized Sprague-Dawley rats of the same sex/age and weight range is ~30 cm H 2 O across studies (Fogarty et al., 2023;Gill et al., 2015;Khurram et al., 2017;Seven et al., 2018).On balance, it appears that P di amplitude sighs are generally equivalent to those generated during airway occlusion.
The post-sigh inter-breath intervals were longer compared to inter-breath intervals for eupnea both PRE-and POST-C 2 SH.In rats, it appears that that the post-sigh respiratory reset is almost immediate, compared to cats, where 5-6 post-sigh breaths remain depressed compared to normal eupnea (Cherniack et al., 1981).An important limitation of the present study is that P di measurements were performed while animals were anesthetized, which largely abolishes the post-inspiratory phase of breathing.It remains to be seen in rats whether the duration of the post-sigh respiratory reset is influenced by post-inspiration DIAm activity.
It is likely that spontaneous sighs utilize or interact with the canonical brainstem respiratory centers (Housley et al., 1970;Ramirez, 2014;Ramirez et al., 2022;Soltysik & Jelen, 2005).The results of the present study indicate that the generation of P di during sighs is sensitive to C 2 SH removal of ipsilateral descending excitatory input to PhMNs.The P di generated POST-C 2 SH are in stark contrast to the P di generated following unilateral phrenicotomy, where P di generated during eupnea and hypoxia/hypercapnia are preserved with an increase in contralateral DIAm EMG, while the P di generated during sigh and airway occlusion are reduced, with no compensatory increase in DIAm EMG on the uninjured side (Gill et al., 2015;Khurram et al., 2017).Following C 4 unilateral contusion, the P di generated during airway occlusion is reduced (Khurram et al., 2019), with no effects on DIAm EMG (Rana et al., 2017).Differences in the effects of C 2 SH and unilateral C 4 contusion are likely related to the differential disruption of descending excitatory inputs to PhMN and the extent of contusion induced PhMN death, for example, with C 2 SH the extent of sparing of ipsilateral versus contralateral inputs to PhMNs is exceedingly important (Fogarty & Sieck, 2020;Rana et al., 2020;Tai & Goshgarian, 1996).With C 4 contusion, frank PhMN death and DIAm denervation occurs on the injured side (Alvarez-Argote et al., 2016;Rana et al., 2017) together with disruption of synaptic inputs to some PhMNs below the lesion.
Overall, this study shows that, in the absence of winnowing out non-"stereotypical" sighs, the sigh is quite variable within and across rats and is highly sensitive to C 2 SH.Likewise the P di of the post-sigh breath is also affected by C 2 SH.In the spinal cord injury population, maintenance of cardiorespiratory health is essential (Fogarty & Sieck, 2020;Raab et al., 2021;Randelman et al., 2021), with sighs highly important in maintaining lung ventilatory function (Housley et al., 1970;Katagiri et al., 1998;Lieske et al., 2000;Ramirez et al., 2022;Riede et al., 2020;Wuyts et al., 2011).Our results of reduced sigh P di amplitude likely impair the effectiveness of this behavior to stave of lung atelectasis.The positive effects of breathing exercises (e.g., breath-stacking) post cervical spinal cord injury may be one way to accomplish what spontaneous sigh cannot in this population (Jeong & Yoo, 2015), although this probably is insufficient to mitigate any risks of pneumonias and airway infections due to impaired coughing (Berlowitz et al., 2016).
Our study also has important consequences for respiratory neurobiology in general, particularly with using sighs as an exemplar for the function of the entire respiratory neuromotor system.We caution against experimental approaches where atypical sighs are arbitrarily excluded and then subsequently used as a normalization, as this may artificially reduce variability, essentially doing nothing other than normalizing to twice eupnea.This is particularly important for conditions where sighs and the post-sigh breaths are particularly vulnerable (e.g., Rett syndrome) (Fogarty, 2023;Huang et al., 2016;Robinson et al., 2012;Voituron et al., 2010).Here, we show that using an inclusive shape-based, not amplitude-based sigh sampling, we are able to capture the true variability within the behavior while also being sensitive enough to see an effect of injury.In the future as evaluations of sigh become less phenomenological, with more attention paid to their genesis within neural circuits, it will be important to pay attention to both the sigh itself and the immediate post-sigh breath, as various species exhibit different P di and temporal relationships to eupnea and maximal DIAm behaviors.

F
I G U R E 1 (a) Representative tracing of P di during eupnea, sigh, and the immediate post-sigh breath in PRE-SHAM, POST-SHAM, PRE-C 2 SH, and POST-C 2 SH conditions.(b) Scatterplot of P di (cm H 2 O) showing reduction in the POST-C 2 SH (orange) compared to the PRE-C 2 SH (green) condition during eupnea, sigh, and the post-sigh breath, with no changes between PRE-SHAM and POST-SHAM with sigh (except during POST-C 2 SH) having greater P di than all other groups (behavior: p < 0.0001; type of surgery: p = 0.0002; and time: p < 0.0001).(c) Scatterplot of normalized P di (P di%PRE ) shows reductions in the POST-C 2 SH compared to PRE-C 2 SH condition during eupnea, sigh, and the post-sigh breath, with no changes between PRE-SHAM and POST-SHAM (behavior: p < 0.0001; type of surgery: p = 0.0002; and time: p < 0.0001).All tests are paired three-way ANOVAs with Bonferroni post-tests, n = 8 for all groups.Each scatter point represents the mean value of each rat, * denotes significant differences within behavior between PRE-C 2 SH and POST-C 2 SH groups for P di during eupnea (p = 0.012), sigh (p < 0.0001) and the post-sigh breath (p = 0.029) and for normalized P di%PRE during eupnea (p = 0.011), sigh (p = 0.0001), and the post-sigh breath (p = 0.010).

F
I G U R E 2 (a) Scatterplot of breath duration (s) showing increased breath duration of sigh compared to eupnea and the immediate post-sigh breath, regardless of injury (behavior: p < 0.0001; type of surgery: p = 0.787; and time: p = 0.227).(b) Scatterplot of breath durations normalized within rats to PRE-injury eupnea values shows extended breath durations during sigh compared to eupnea and the post-sigh breath, regardless of injury (behavior: p < 0.0001; type of surgery: p = 0.469; and time: p = 0.270).All tests are paired three-way ANOVAs with Bonferroni post-tests, n = 8 for all groups.Each scatter point represents the mean value of each rat.F I G U R E 3 (a) Scatterplot of inter-breath intervals (s) showing increased inter-breath intervals (IBE) of the post-sigh breath compared to all other behaviors, regardless of injury (behavior: p < 0.0001; type of surgery: p = 0.659; and time: p = 0.610).(b) Scatterplot of IBE normalized within rats to PRE eupnea values shows longer inter-breath intervals in the post-sigh breath regardless of injury (behavior: p < 0.0001; type of surgery: p = 0.924; and time: p = 0.661).All tests are paired three-way ANOVAs, n = 8 for all groups.Each scatter point represents the mean value of each rat.

F
I G U R E 4 (a) Scatterplot showing the % of all sighs exhibiting "stereotypical" twice eupnea P di shows reduction in the % of sighs with canonical P di amplitudes in POST-C 2 SH compared to PRE-C 2 SH rats (p = 0.0078, Wilcoxon matched-pairs signed-rank test).(b) Scatterplot showing the % of all sighs exhibiting "stereotypical" twice eupnea inter-breath intervals (i.e., post-sigh respiratory reset) of the post-sigh breath shows no change with injury in the % of sighs with canonical post-sigh respiratory reset in PRE-and POST-C 2 SH groups (p = 0.373, paired t-test).Each scatter point represents the mean value of each rat, n = 8 for all groups, * denotes significant differences (p = 0.0078) within behavior between PRE-and POST-C 2 SH groups.

F
I G U R E 5 (a) Scatterplot of all individual sigh P di (cm H 2 O), showing reduced amplitudes in the POST-C 2 SH compared to PRE-C 2 SH timepoints (p < 0.0001).(b) Scatterplot of all individual inter-breath intervals (s) between sigh and the post-sigh breath, showing no difference between PRE-and POST-C 2 SH timepoints (p = 0.979).In all plots, green symbols represent "stereotypical" sighs of >twice eupneic P di and post-sigh respiratory reset, purple symbols represent sighs satisfying "stereotypical" P di criteria, yellow symbols represent sighs satisfying inter-breath interval criteria and red symbols represent sighs where neither criterion was satisfied.All tests are unpaired t-tests, * denotes p < 0.0001, n = 36 (PRE-C 2 SH) and n = 24 (POST-C 2 SH).
Number and percentages of sighs exhibiting stereotypical criteria in PRE-C 2 SH and POST-C 2 SH conditions.Italicized POST values are significantly different from PRE, Fischer's exact test, p < 0.05.
Variance and effect sizes between the PRE-C 2 SH and POST-C 2 SH states across rat means.